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Zeitschrift/Journal: European Journal of

Jahr/Year: 2017

Band/Volume: 0286

Autor(en)/Author(s): Sabroux Romain, Corbari Laure, Krapp Franz, Bonillo Celine, Le Prieur Stephanie, Hassanin Alexandre

Artikel/Article: Biodiversity and phylogeny of (Arthropoda: Pycnogonida) 1-33 European Journal of Taxonomy 286: 1–33 ISSN 2118-9773 http://dx.doi.org/10.5852/ejt.2017.286 www.europeanjournaloftaxonomy.eu 2017 · Sabroux R. et al. This work is licensed under a Creative Commons Attribution 3.0 License.

DNA Library of Life, research article

urn:lsid:zoobank.org:pub:8B9DADD0-415E-4120-A10E-8A3411C1C1A4

Biodiversity and phylogeny of Ammotheidae (Arthropoda: Pycnogonida)

Romain SABROUX 1, Laure CORBARI 2, Franz KRAPP 3, Céline BONILLO 4, Stépahnie LE PRIEUR 5 & Alexandre HASSANIN 6,* 1,2,6 UMR 7205, Institut de Systématique, Evolution et Biodiversité, Département Systématique et Evolution, Sorbonne Universités, Muséum national d’Histoire naturelle, 55 rue Buffon, CP 51, 75005 Paris, France. 3 Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, 53113 Bonn, Germany. 4,5 UMS CNRS 2700, Muséum national d’Histoire naturelle, CP 26, 57 rue Cuvier, 75231 Paris Cedex 05, France.

* Corresponding author: [email protected] 1 Email: [email protected] 2 Email: [email protected] 3 Email: [email protected] 4 Email: [email protected] 5 Email: [email protected]

1 urn:lsid:zoobank.org:author:F48B4ABE-06BD-41B1-B856-A12BE97F9653 2 urn:lsid:zoobank.org:author:9E5EBA7B-C2F2-4F30-9FD5-1A0E49924F13 3 urn:lsid:zoobank.org:author:331AD231-A810-42F9-AF8A-DDC319AA351A 4 urn:lsid:zoobank.org:author:7333D242-0714-41D7-B2DB-6804F8064B13 5 urn:lsid:zoobank.org:author:5C9F4E71-9D73-459F-BABA-7495853B1981 6 urn:lsid:zoobank.org:author:0DCC3E08-B2BA-4A2C-ADA5-1A256F24DAA1

Abstract. The family Ammotheidae is the most diversifi ed group of the class Pycnogonida, with 297 described in 20 genera. Its monophyly and intergeneric relationships have been highly debated in previous studies. Here, we investigated the phylogeny of Ammotheidae using specimens from poorly studied areas. We sequenced the mitochondrial gene encoding the fi rst subunit of cytochrome c oxidase (CO1) from 104 specimens. The complete nuclear 18S rRNA gene was sequenced from a selection of 80 taxa to provide further phylogenetic signal. The base composition in CO1 shows a higher heterogeneity in Ammotheidae than in other families, which may explain their apparent polyphyly in the CO1 tree. Although deeper nodes of the tree receive no statistical support, Ammotheidae was found to be monophyletic and divided into two clades, here defi ned as distinct subfamilies: Achelinae comprises the genera Hodge, 1864, Ammothella Verrill, 1900, Haswell, 1884 and Miers, 1879; and Ammotheinae includes the genera Ammothea Leach, 1814, Acheliana Arnaud, 1971, Cilunculus Loman, 1908, Fry & Hedgpeth, 1969 and also Teratonotum gen. nov., including so far only the type species Ammothella stauromata Child, 1982. The species Cilunculus gracilis Nakamura & Child, 1991 is reassigned to Ammothella, forming the binomen Ammothella gracilis (Nakamura & Child, 1991) comb. nov. Additional taxonomic re-arrangements are suggested for the genera Achelia, Acheliana, Ammothella and Cilunculus.

1 European Journal of Taxonomy 286: 1–33 (2017)

Keywords. Pantopoda, taxonomy, DNA phylogeny, barcode of life, strand-bias.

Sabroux R., Corbari L., Krapp F., Bonillo C., Le Prieur S. & Hassanin A. 2017. Biodiversity and phylogeny of Ammotheidae (Arthropoda: Pycnogonida). European Journal of Taxonomy 286: 1 –33. http://dx.doi.org/10.5852/ ejt.2017.286

Introduction Sea spiders (Arthropoda: Pycnogonida: Pantopoda) represent a small group of exclusively marine which are distributed worldwide, from the tropical to the polar regions, from littoral to abyssal depths. There are 1385 described species that are classifi ed in 79 genera and 11 families (Bamber et al. 2015). They exhibit a typical “spider-like” appearance, with generally four pairs of walking legs attached to a slender trunk. They show a remarkable diversity of forms (from the slender Fabricius, 1794 to the stout Pycnogonum Brünnich, 1764), a variable number of leg pairs (from 4 to 6), a wide size range (leg span from 1 to 700 mm) and a great diversity of colours, ranging from the palest to the most colourful (e.g., the multi-coloured Anoplodactylus evansi Clark, 1963) (Arnaud & Bamber 1987; Bamber et al. 2015). The position of sea spiders as a class of the subphylum is accepted by most recent molecular studies (Regier et al. 2010; Rehm et al. 2014; Roeding et al. 2009). The pycnogonid families were distinguished based on the presence/absence and the structure of the three cephalic appendages: chelifores, palps and ovigers (e.g., Hedgpeth 1948). However, the number of families has varied heavily during the taxonomic history of Pycnogonida, from 8 families in Hedgpeth (1948) to 27 in Fry (1978). More recent classifi cations have recognized between 9 (Arnaud & Bamber 1987) and 11 families (Bamber 2007b). The position of some genera was also highly debated (e.g., Pallenopsis Wilson, 1881, Endeis Philippi, 1843, Tanystylum Miers, 1879). The four previous molecular studies on pycnogonids have questioned the validity of several families (Arango 2003b; Arango & Wheeler 2007; Nakamura et al. 2007; Arabi et al. 2010). Among them, the most problematic taxon probably remains the family Ammotheidae. Originally, Dohrn (1881) gave an extensive defi nition of Ammotheidae by integrating the genera Ammothea Leach, 1814, Barana Dohrn, 1881 (currently accepted as Ascorhynchus Sars, 1878), Clotenia Dohrn, 1881 (accepted as Tanystylum) and Trygaeus Dohrn, 1881. Later during the same year, Hoek (1881) created the family Ascorhynchidae combining Ascorhynchus, Ammothea and Tanystylum, inter alia. The question of splitting Ammothea and Ascorhynchus (and their respective relatives) into two families has always been a matter of debate, because they show puzzling combinations of characters (e.g., the same chelae reduction vs different oviger structures). While most specialists included them in a single family (e.g., Arnaud & Bamber 1987; Dohrn 1881; Hedgpeth 1941; Hoek 1881; Nakamura & Child 1991; Stephensen 1933; Stock 1994), some early authors split them into two families (e.g., Bouvier 1923). Finally, molecular studies (Arango & Wheeler 2007; Nakamura et al. 2007; Arabi et al. 2010) cast doubt on the hypothesis of a single family (which was Ammotheidae according the priority rule of taxonomic nomenclature). Nakamura et al. (2007) suggested the resurrection of the family Ascorhynchidae, a position that was followed by the commonly used classifi cations of Bamber (2007b; 2015), but Ascorhynchidae and Ammotheidae were grouped into the same superfamily Ascorhynchoidea Hoek, 1881 (and not “Pocock, 1904” as listed by Bamber 2007b and Bamber et al. 2015). Even now, the status of Ammotheidae sensu Bouvier (1923) (i.e., excluding Ascorhynchidae) is rather unclear. As previously commented by Bouvier, most of the characters used to describe this reduced taxon show exceptions: for instance, their main character, i.e., the reduction of chelae to small buds, is not constant (for example, adults of Nymphopsis muscosa Loman, 1908 bear chelate chelae); the number of palp articles is highly variable (4 to 9); the abdomen can be articulated or not to the trunk, and their development can be direct or larval (Bamber 2007b). The family Ammotheidae sensu Bouvier (1923)

2 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae was found to be poly- or paraphyletic in the molecular studies of Nakamura et al. (2007) and Arango & Wheeler (2007). However, these results may have been caused by the use of unreliable molecular data (carryover DNA contamination and high levels of missing data, inaccurate methods of DNA alignment and the extreme heterogeneity of nucleotide composition in the mitochondrial genes of sea spiders, see Arabi et al. 2010). Nevertheless, the taxon was recovered monophyletic in the tree obtained from a concatenation of fi ve mitochondrial and nuclear markers, but based on only ten species (of the 297 described) (Arabi et al. 2010). Therefore, we consider that the status of Ammotheidae is an open question that needs to be studied with a better taxonomic sampling, including more species diversity. Previous studies on sea spiders have mainly focused on species from the Southern Ocean. As a consequence, we have a great amount of knowledge on this fauna in terms of biodiversity (León 2001; Munilla & Soler-Membrives 2009, 2015), integrative taxonomy, population genetics (Arango et al. 2011; Dietz et al. 2015; Krabbe et al. 2010), biogeography (Griffi ths et al. 2011; Munilla & Soler- Membrives 2009) and parasitology (Schiaparelli et al. 2008). Historically, one of the fi rst invertebrate species described from Antarctica was a (Decolopoda australis Eights, 1835) and since then, pycnogonids have been considered as a fl agship group in Antarctica. Moreover, taxonomists were particularly interested in Antarctic sea spiders because of their large size and their “extra-legged” representatives (ten legs or more) (e.g., Bouvier 1910). In contrast, species from non-Antarctic regions are often tiny in size, and thus more diffi cult to collect or study by non-specialists. As a consequence, most of the barcode sequences (5’ fragment of the gene encoding the fi rst subunit of cytochrome c oxidase, CO1) currently available for Ammotheidae in the nucleotide databases come from southern ecoregions (South Australia, South America and Antarctica; Fig. 1). Here, we investigated the diversity and phylogeny of Ammotheidae by focusing on sea spiders collected in several poorly studied tropical areas (e.g., Papua New Guinea, South Madagascar, Marquesas Islands) during the latest expeditions of the Muséum national d’Histoire naturelle of Paris (MNHN). We generated 104 sequences of the CO1 mitochondrial gene and 80 sequences of the 18S rRNA (18S) nuclear gene. These datasets were analysed to address the following three main questions: (1) Are Ammotheidae monophyletic? (2) Are the “big fi ve” ammotheid genera (Ammothea, Achelia Hodge, 1864, Ammothella Verrill, 1900, Cilunculus Loman, 1908 and Tanystylum) monophyletic? (3) How heterogeneous is the base composition in CO1 sequences of Ammotheidae? Material and methods Sampling The specimens selected for this study were collected during the following MNHN expeditions and deep sea cruises (Fig. 2), organized under the “Planète Revisitée” and “Tropical Deep Sea Benthos” programs (Bouchet et al. 2008; Richer de Forges et al. 2013): BATHUS 3 (New Caledonia, 1993), SANTO (Vanuatu, 2006), CEAMARC (Antarctica, 2008), MAINBAZA (Mozambique Channel, 2009), ATIMO VATAE (South Madagascar, 2010), BIOPAPUA (Bismarck Sea, Papua New Guinea, 2010), PAPUA NIUGINI (Madang, Papua New Guinea, 2012), PAKAIHI I TE MOANA (Marquesas Islands, 2012), KARUBENTHOS (Guadeloupe, 2012); KAVIENG (Kavieng, Papua New Guinea, 2014), GUYANE 2014 (off French Guiana, 2014), and ILES DU SALUT (French Guiana, 2014). Details of the fi eld operations are available at http://expeditions.mnhn.fr/. Some additional specimens have been provided by IFREMER (BICOSE, BIOBAZ, HYDROMAR and FUTUNA 3 cruises). All the geographical locations are reported in Appendix 1. The specimens were collected from littoral shores to hydrothermal vents (3500 m depth).

DNA extraction, amplifi cation and sequencing Total DNA was extracted from a leg removed on specimens preserved in 80–95% ethanol using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany). The samples were lysed in 360 μl of ATL (twice

3 European Journal of Taxonomy 286: 1–33 (2017) as much as recommended by the manufacturer) and 40 μl of proteinase K. The volumes of AL and ethanol were also doubled. The rest of the protocol followed the volumes indicated by the manufacturer. Final extract volumes contained between 25 and 100 μl of DNA solution.

Two markers were sequenced for this study: the mitochondrial CO1 gene and the nuclear 18S gene. We used a new set of primers to amplify CO1 sequences (Py-CO1-U: 5’-TCA-ACW-AAT-CAT-AAA-GAY- ATT-GG-3’ and Py-CO1-L3: 5’-GGR-TCH-CCH-CCH-GMD-GGR-TC-3’) and the three sets used in previous studies for the 18S sequences (see details in Arabi et al. 2010). DNA amplifi cation were done using Hot start mix RTG Taq (GE Healthcare, Waukesha, WI, USA) in a 25 μl fi nal volume containing between 1 and 5 μl of DNA and 1μl of each 10 mM primer. Initial denaturation was performed at 94°C for 4 min, then we applied 40 cycles of denaturation-hybridization of 30 s at 94°C, 30 s at hybridization temperature (50°C for CO1, 52°C for 18S), and 1 min at 72°C. Final elongation lasts 10 min at 72°C. Purifi cation and cycle sequencing were performed by Eurofi ns (Munich, Germany) using the PCR primers detailed above.

Fig. 1. Barcode richness in GenBank before and after this study. A–B. Number of CO1 haplotypes before (A) and after (B) this study as a function of the origins of the specimens sequenced. C. Number of species (grey) and genera (black) represented by a CO1 sequence found in GenBank databases before (left) and after (right) this study, classifi ed by ecoregions. Littoral ecoregions defi ned according to Spalding et al. (2007) and abyssal ecoregions following a simplifi cation of Bachraty et al. (2009).

4 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

The sequences were cleaned using CodonCode Aligner v. 3.7.1 (CodonCode Corporation, Dedham, MA, USA) by comparing forward and reverse electrophoregrams. Nucleotide ambiguities were coded according to the IUPAC nomenclature. Cleaning was achieved after multiple DNA alignments using Se-Al v. 2.0a11 (Rambaut 2002). Each sequence was compared via BLAST methods (NCBI, Benson et al. 2015) in order to detect potential contaminations. The sequences were deposited in GenBank under accession numbers KX535346–KX535450 (CO1) and KX536422–KX536501 (18S).

Phylogenetic analyses The 104 CO1 barcodes and 80 18S sequences generated for this study were compared to all sequences of Ammotheidae (48) and Aschorhynchidae (19) downloaded from GenBank. For other pycnogonid families, we selected only species for which both CO1 and 18S sequences were available. Details on the 75 CO1 and 79 18S sequences from GenBank are provided in Appendix 1. The pycnogonid tree was rooted with 10 outgroup species, including two chelicerates (Uropygi: Mastigoproctus giganteus (Lucas, 1835); Xiphosura: Limulus polyphemus (Linnaeus, 1785)), six mandibulates (Stomatopoda: Squilla empusa Say, 1818; Branchiopoda: Triops longicaudatus LeConte, 1846; Chilopoda: Lithobius variegatus Leach, 1814 and L. forfi catus (Linnaeus, 1785); Diplopoda: Thyropygus sp. and Orthoporus sp.) and two onychophorans (Opisthopatus cinctipes Purcell, 1899 and Peripatoides novaezaelandiae (Hutton, 1876)). We avoided CO1 sequences characterized by an inverted bias in base composition (e.g., scorpions, spiders, etc.) in order to limit artefacts during phylogenetic reconstruction. This strategy was determined in agreement with previous studies showing that asymmetric mutational constraints occurred during the evolution of the mitochondrial genome of Chelicerata, including sea spiders (Hassanin et al. 2005; Hassanin 2006; Arabi et al. 2010, 2012).

Fig. 2. Regional distribution of the studied specimens. Green diamonds indicate locations where specimens were collected at shallow depths (< 100 m), blue circles those from deep-sea samplings; the combinations of both symbols indicates mixed samplings (below and above 100 m depth). 1: Futuna; 2: Marquesas Islands; 3: Guadeloupe; 4: French Guiana; 5: Mid-Atlantic Ridge, Snake Pit site; 6: Mid- Atlantic Ridge, Logatchev site; 7: Mid-Atlantic Ridge, Lucky Strike site; 8: Mozambique Channel; 9: South Madagascar; 10: Antarctica, Terre Adélie; 11: Papua New Guinea; 12: New Caledonia and Vanuatu.

5 European Journal of Taxonomy 286: 1–33 (2017)

The 18S alignment was achieved using a fi rst trial with CodonCode Aligner v. 3.7.1, and it was optimized manually on Se-Al v. 2.0a11. Several regions with too many ambiguous positions for homology were removed from the analyses. In other regions that are diffi cult to align, but which provide phylogenetic information at lower taxonomic levels, we chose a different strategy based on the use of different taxonomic blocks. For instance, the region located at positions 679–693 (with respect to the DQ389932 sequence) was aligned using four shifted blocks corresponding to the following taxa: (1) Ammotheidae Dohrn, 1881, Nymphonidae Wilson, 1878, Callipallenidae Hilton, 1942 and Pallenopsidae Fry, 1978, (2) Ascorhynchidae Hoek, 1881 and Endeidae Norman, 1908, (3) Phoxichilidiidae Sars, 1891, and (4) Jarvinsky, 1870, Pycnogonidae Wilson, 1878 and Austrodecidae Stock, 1954.

Phylogenetic analyses were performed with MrBayes v. 3.2.2 (Ronquist & Huelsenbeck 2003), running 4 chains for 10 million generations, and a 25% burn-in. We analysed the 18S and CO1 (partitioned by codon positions or without partition) datasets separately, and the model (i.e., GTR + G + I) was selected using the best AIC score calculated on jModelTest (Posada 2008). For the reduced dataset of 135 taxa used for the concatenation of CO1 and 18S markers, we performed a partitioned analysis using a GTR for each marker.

Analysis of the nucleotide compostion in COI sequences Strand-bias in nucleotide composition was analysed at third codon positions of CO1 sequences. As similar trends were previously found for two- and fourfold degenerate sites (Hassanin et al. 2005), we followed the simplifi ed approach previously published in Arabi et al. (2010), in which AT and CG skews were calculated on all third codon positions of CO1 sequences using the following formulas:

AT skew = (FA - FT) / (FA + FT) and CG skew = (FC - FG) / (FC + FG), where F is the frequency of the considered nucleotide at third codon positions. The skew values were considered as signifi cant if the null hypothesis could be rejected to a confi dence level of 5%.

Results Datasets The great extent of localities and depth range explored here allowed us to examine a large diversity of pycnogonids from 8 of the 11 families and from the seven main genera of Ammotheidae, except Ammothea (i.e., Achelia, Ammothella, Cilunculus, Nymphopsis Haswell, 1884, Sericosura Fry & Hedgpeth, 1969 and Tanystylum). The presently monospecifi c Acheliana Arnaud, 1971 is also sequenced for the fi rst time with an undescribed species (in prep.). With the sequences extracted from GenBank (see Appendix 1), we included one additional ammotheid (Ammothea) and two other families (Austrodecidae and Colossendeidae) in the analyses.

We detected several potential errors in the GenBank sequences, involving substitutions in highly conserved regions (e.g., Ammothea hilgendorfi DQ389936; Anoplodactylus batangensis DQ389918), as well as unexpected indels in the 18S stems, which are very constrained regions (see below) (e.g., Ascorhynchus castelloides DQ389905; novaezealandiae DQ389927; Anoplodactylus batangensis DQ389918). However, these potential errors were not so problematic for phylogenetic inferences, since most of them are autapomorphic. More worrying is the misleading effect of the 18S sequence of Pentapycnon charcoti (DQ389924) on phylogenetic reconstruction. Indeed, our preliminary analyses revealed its chimeric origin as positions 1 to 923 correspond to an undetermined fungus. In this case, we excluded the fungal part of the sequence from the alignment. Similar problems have previously been described in Arabi et al. (2010) for other sea spiders. Other sequences susceptible to generate reconstruction artefacts, due to their poor quality (highly divergent sequences in conserved regions) or their shortness, were removed from the analyses (CO1: Achelia alaskensis DQ390093; 18S: Pallenopsis macronyx DQ389908).

6 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

The CO1 alignment contains 179 sequences (of which 104 are new) and 618 nucleotides representing 376 informative characters (369 without outgroups). It is worth noting that all specimens of the genus Eurycyde Schiödte, 1857 share a synapomorphic deletion of two codons in the CO1 gene (positions 472–477 in the DQ390087 sequence of Achelia assimilis). There is no signifi cant difference between the CO1 analyses made with or without partition (Fig. 3). The 18S alignment contains 159 sequences (of which 80 are new) and 1750 nucleotides (including gaps due to alignment) representing 321 informative characters (idem without outgroups). The 18S tree is shown in Fig. 4. The concatenation of the two markers contains 135 sequences and 2550 nucleotides representing 746 informative characters (602 without outgroups). The tree obtained from the concatenation is illustrated in Fig. 5.

Phylogenetic relationships The monophyly of Pycnogonida is supported by maximal values of posterior probability (PP = 1) in all analyses. Most families are monophyletic with high support (PP > 0.9) in most analyses: Colossendeidae, Endeidae, Pallenopsidae, Phoxichilidiidae and Pycnogonidae. In the CO1 tree (Fig. 3), however, Pallenopsidae is recovered as paraphyletic (PP = 0.87) and the monophyly of Phoxichilidiidae is less robust (PP < 0.5). The family Ascorhynchidae is found to be monophyletic in the concatenated analysis (PP = 0.9) (Fig. 5) and the 18S analysis based on 135 specimens (PP = 0.72) (Appendix 2), whereas different, but unrobust relationships are shown in other analyses (PP < 0.6). All members of the families Callipallenidae and Nymphonidae are systematically grouped, but these taxa are always found to be either poly- or paraphyletic.

Within pycnogonids, most basal relationships are poorly supported (PP < 0.8), but the families Austro- decidae and Pycnogonidae are found to be the fi rst divergent lineages in most topologies (Figs 3–5, Appendix 2). However, these results only show good support in the combined analysis, where Austrodecidae and Pycnogonidae are the fi rst and second offshoots, respectively (PP > 0.85). The family Colossendeidae is the next taxon to diverge in both combined and 18S analyses, but this has only weak support (PP = 0.57–0.77) (Figs 4–5). In the CO1 analyses (Fig. 3, Appendix 2), Colossendeidae appear as the sister-group of Phoxichilidiidae (PP = 0.69 and 0.93), and they are allied to the clade uniting Nymphonidae and Callipallenidae (PP = 0.82) in the CO1 analysis based on 179 haplotypes (Fig. 3).

The clade uniting Callipallenidae and Nymphonidae is grouped with Ascorhynchidae in the 18S and combined analyses (PP = 0.72–0.93) (Figs 4–5, Appendix 2). In the CO1 analysis based on 135 specimens (Appendix 2), however, the paraphyletic ascorhynchids are grouped with some ammotheids (i.e., excluding the “Achelia sawayai group”, see below) and Pallenopsidae (PP = 0.81).

The family Ammotheidae appears to be monophyletic in the 18S and CO1 + 18S trees with good supports (PP = 0.83–0.95) (Figs 4–5, Appendix 2). The CO1 analyses (Fig. 3, Appendix 2), however, support its polyphyly (PP = 0.81–0.9) due to the position of the “Achelia sawayai group” (Achelia sawayai Marcus, 1940 + Achelia sp. 15 + specimen MNHN-IU-2013-18602), which is found to be monophyletic with maximum support in all analyses (PP = 1) and divergent from other Ammotheidae (PP = 0.79).

Two major subfamilies of Ammotheidae can be distinguished: (1) Achelinae Wilson, 1881, which includes Achelia, Nymphopsis, Tanystylum, all Ammothella except A. stauromata Child, 1982 and A. biunguiculata Dohrn, 1881, and Cilunculus gracilis Nakamura & Child, 1991 (see 18S analysis in Fig. 4); and (2) Ammotheinae Dohrn, 1881 emend., which is composed of the genera Ammothea, Acheliana, Sericosura, all Cilunculus but C. gracilis, and the species Ammothella stauromata and A. biunguiculata (see 18S analysis in Fig. 4). The subfamily Ammotheinae is supported in all analyses, in general with highest support values. The subfamily Achelinae is monophyletic in the 18S and CO1 + 18S trees (PP = 1) (Figs 4–5, Appendix 2), but paraphyletic in the CO1 trees (PP = 0.94–0.98) (Fig. 3, Appendix 2) because the “Achelia sawayai group” appears as the sister-group of Endeidae

7 European Journal of Taxonomy 286: 1–33 (2017)

Fig. 3. Bayesian tree of Pycnogonida based on 179 sequences of the mitochondrial CO1 gene (partitioned analysis). Coloured rectangles show non-ammotheid families, and coloured branches discriminate ammotheid genera. The numbers at the nodes indicate posterior probabilities greater than 0.5. Symbols associated with each taxon name indicate the bias in base composition, as expressed by AT (circles) and CG (squares) skews (see main text for details): blue symbols represent a signifi cant positive bias; red symbols indicate a signifi cant negative bias; uncoloured symbols show insignifi cant values of skews. Asterisks after taxon names indicate holotype specimens. The arrow at the top of the tree shows the connection with Part 2 of the tree (see next page).

8 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

Fig. 3. Part 2, see Part 1 for explanation.

9 European Journal of Taxonomy 286: 1–33 (2017)

Fig. 4. Bayesian tree of Pycnogonida based on 159 sequences of the nuclear 18S rRNA gene. Coloured rectangles show non-ammotheid families and coloured branches discriminate ammotheid genera. The numbers at the nodes indicate posterior probabilities greater than 0.5. Asterisks after taxon names indicate holotype specimens. Outgroups were removed for better readability. The arrow at the top of the tree shows the connection with Part 2 of the tree (see next page).

10 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

Fig. 4. Part 2, see Part 1 for explanation.

11 European Journal of Taxonomy 286: 1–33 (2017)

(PP = 0.5–0.74). In the 18S and combined analyses (Figs 4–5, Appendix 2), the “Achelia sawayai group” is included within Achelinae, where it constitutes a strongly supported clade with Tanystylum (PP = 1). Within Achelinae, the monophyly of Nymphopsis is recovered in all analyses (PP = 0.56–0.64 in CO1 analyses, PP = 1 in 18S and concatenated analyses). In the CO1 + 18S tree (Fig. 5), Ammothella s. str. (excluding A. stauromata and A. biunguiculata) constitutes a monophyletic clade (PP = 1), but the 18S tree (Fig. 4) shows that Cilunculus gracilis (which is absent from the CO1 tree) also falls into this clade (PP = 0.95). In the CO1 analyses (Fig. 3, Appendix 2), Ammothella s. str. is found to be polyphyletic: a fi rst group, which includes Ammothella sp. 7, A. tuberculata Cole, 1904 and A. appendiculata (Dohrn, 1881), is related to Nymphopsis with rather strong support (PP = 0.6–0.88); a second group, which is only represented by the species Ammothella setacea (Helfer, 1938), is close to the genus Tanystylum (PP = 0.9); and a third group, which is composed of Ammothella sp. 1, 2, 6, 12, 14, 15, A. spinifera Cole, 1904 and A. tippula Child, 1983 (PP = 0.6), shows an unstable position with respect to other lineages of Achelinae. The monophyly of Achelia s. str. (i.e., excluding the “Achelia sawayai group”) is well supported in all analyses (PP = 0.92–1), except in the 18S tree of Fig. 5, where its paraphyly is not found to be robust (PP < 0.5). Within Ammotheinae, Ammothea is constantly shown as paraphyletic due to the inclusive position of the genus Acheliana (PP = 1) (Figs 3–5, Appendix 2). The genus Cilunculus (excluding C. gracilis) is found to be monophyletic in the 18S and CO1 + 18S trees (PP = 0.95–1). The genus Sericosura is monophyletic in all analyses (PP = 0.97–1).

Nucleotide composition in CO1 sequences The analysis of third codon positions of the CO1 gene shows that most families of Pycnogonida are characterized by positive values for AT and CG skews (Fig. 3; detailed values in Supplementary fi le), which means that their sequences have an excess of A relative to T nucleotides and of C relative to G nucleotides. However, the AT skew is signifi cantly negative in most species of the genus Anoplodactylus Wilson, 1878 (10 on 14), and in Austrodecus glaciale Hodgson, 1907. In the clade grouping Callipallenidae and Nymphonidae, two unrelated species (MNHN-IU-2014-8371 Nymphonidae gen. sp. and Nymphon hamatum) also show negative AT and CG skews. For the family Ammotheidae, our analyses revealed a higher heterogeneity of base composition. The members of the subfamily Achelinae exhibit a strong heterogeneity: for most taxa, the skew values are non-signifi cant (e.g., Nymphopsis and Achelia excluding the “Achelia sawayai group”); Ammothella, Achelia and Tanystylum generally have negative skews, but with some exceptions (Achelia boschi Stock, 1992; A. assimilis (Haswell, 1885); Ammothella spinifera Cole, 1904; Ammothella sp. 15; Tanystylum neorhetum Marcus, 1940 all show positive skews); the “Achelia sawayai group,” however, shows a strong positive bias. Discussion Monophyly and low supports of interfamilial relationships Both our CO1 and 18S analyses supported the monophyly of most pycnogonid families. In contrast, the two families Ammotheidae and Ascorhynchidae were only recovered as monophyletic in the 18S trees. In the CO1 trees, the polyphyly of Ammotheidae sensu Bouvier (1923) seems to be robust (but see next paragraph below), whereas the polyphyly of Ascorhynchidae is not highly supported (PP < 0.6). For Ascorhynchidae, the lack of robustness suggests that their basal diversifi cation (i.e., the divergence between Ascorhynchus and Eurycyde) was more ancient than in other families, excepting, perhaps, Ammotheidae (see below). Indeed, for the deepest nodes of the CO1 trees most of the genuine phylogenetic signal has been erased because of the high saturation of synonymous substitutions in the mitochondrial genome and the low levels of variation observed at non-synonymous sites of the CO1 gene (very high selective pressure). This explains why the CO1 gene did not provide robust support for most interfamilial relationships (Fig. 5). The sole exception concerns Callipallenidae and Nymphonidae,

12 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

Fig. 5. Bayesian tree of Pycnogonida obtained from the concatenation of CO1 and 18S genes (135 taxa). Coloured rectangles show non- ammotheid families and coloured branches discriminate ammotheid genera. The numbers at the nodes indicate posterior probabilities greater than 0.5. Bold branches indicate CO1 (yellow), 18S (blue), or both (red) support in the independent analyses of CO1 and 18S genes provided in Appendix 1. Asterisks after taxon names indicate holotype specimens. Outgroups were removed for better readability.

13 European Journal of Taxonomy 286: 1–33 (2017) which are grouped together at the end of a very long branch in the CO1 and CO1 + 18S trees (Figs 3, 5). This result clearly indicates that the divergence between Callipallenidae and Nymphonidae is more recent than that of other families. In agreement with this view, they share many morphological characters, including the structure of the 10-articled ovigers and the presence of well-developed chelae (Bamber 2007b). In addition, our datasets did not provide any evidence for their reciprocal monophyly. On the contrary, our analyses suggested that these two families are reciprocally polyphyletic (PP < 0.92). However, we found very different polyphyletic patterns in the combined, CO1 and 18S analyses (Figs 3–5), which suggests that CO1 and/or 18S genes are not the best markers to resolve relationships at this level of the pycnogonid tree (Figs 3–4, Appendix 2).

Even with the 18S dataset, most basal relationships within Pycnogonida were not well-resolved, suggesting that most families of Pycnogonida have diverged rapidly from each other. As pointed out by Charbonnier et al. (2007), there is a substantial hiatus in the fossil record of Pycnogonida, between the (ca 400 Mya) and the (ca 160 Mya), and the concomitant appearance of several different families during the Mesozoic is in agreement with a crown group radiation. To explain both the lack of resolution for interfamilial relationships and the long branch that separates outgroups from pycnogonids, Arabi et al. (2010) have also suggested that a very long period of time occurred between the origin of Pycnogonida, and the subsequent rapid diversifi cation that led to extant families.

Strong heterogeneity in base composition in the CO1 gene of Ammotheidae The analyses of CO1 and 18S genes revealed real discordance regarding the phylogeny of Ammotheidae. In the 18S tree (Fig. 4), the family Ammotheidae is found to be monophyletic and it can be divided into two major clades corresponding to the subfamilies Ammotheinae and Achelinae. In the CO1 tree (Fig. 3), the family appears to be polyphyletic: most species of Achelinae fall into the paraphyletic Pallenopsidae, whereas those of the “Achelia sawayai group” are related to the Endeidae, the Ammotheinae being their sister group. As exposed in detail below, we suggest that the apparent topological confl ict between CO1 and 18S markers is the consequence of the combined effects of mutational saturation and multiple changes in base composition during the mtDNA evolution of Ammotheidae.

Previous studies have shown that in most species of Metazoa, the double-stranded and circular mitochondrial genome presents a typical strand asymmetry, in which synonymous sites of the positive strand are characterized by an excess of A relative to T nucleotides and of C relative to G nucleotides (i.e., positive AT and CG skews), while synonymous sites of the complementary negative strand show a reverse bias. This strand-bias in base composition is the consequence of asymmetric mutational constraints during replication and/or transcription of the mtDNA genome (Hassanin et al. 2005; Hassanin 2006). Hassanin et al. (2005) have suggested that the orientation of the control region of the mtDNA is crucial in the establishment of asymmetric mutational constraints, because this region contains both replication and transcription origins. In support of that hypothesis, they showed that two kinds of mitogenomic inversions can lead to a reversal in nucleotide composition: (1) inversion of the control region can result in a global reversal of asymmetric mutational constraints; (2) inversion of a genomic fragment can result in a local reversal of asymmetric mutational constraints. Several reversals of strand specifi c bias were identifi ed in CO1 sequences of Chelicerata (Arabi et al. 2010, 2012) including the common ancestors of Scorpiones and Opisthothelae spiders, as well as several taxa within Acari, Opiliones, Pseudoscorpiones and Pycnogonida. Within Pycnogonida, Arabi et al. (2010, 2012) have revealed a strong heterogeneity in base composition, particularly among ammotheids, with taxa characterized by positive AT and CG skews (Achelia assimilis), positive CG skew but no signifi cant bias for AT (Ammothea), negative AT and CG skews (e.g., Achelia hispida Hodge, 1864, Ammothella tuberculata and Nymphopsis duodorsospinosa Hilton, 1942) and negative AT skew but no signifi cant bias for CG (Ammothella appendiculata). Our analyses showed that the situation is even more complex with the detection of two additional categories: taxa with no signifi cant strand-bias (e.g., Nymphopsis)

14 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae and taxa with negative CG skew but no signifi cant bias for AT (e.g., Tanystylum orbiculare Wilson, 1878). More importantly, our analyses of base composition in CO1 sequences suggest that the apparent polyphyly of Ammotheidae and that of Achelinae resulted from artefacts in tree reconstruction, because Ammotheinae, Achelinae s. str. (excluding the “Achelia sawayai group”) and the “Achelia sawayai group” exhibit clear differences in base composition. Indeed, many species of Achelinae excluding the “Achelia sawayai group” are characterized by one or two negative skews in their CO1 sequence. Only four species of this large clade appear to be characterized by positive AT and CG skews (Achelia assimilis, Ammothella sp. 15 + A. spinifera, and Tanystylum neorhetum), and all of them occupy a derived position within the subfamily. As a consequence, we can infer that the CO1 sequence of the most recent common ancestor of Achelinae was characterized by a negative AT skew and a negative or non-signifi cant CG skew. In contrast, all species of the “Achelia sawayai group” clade have positive AT and CG skews, and those of the Ammotheinae clade generally show a positive CG skew associated with a non-signifi cant AT skew. As pointed out in previous studies, important differences in base composition of mtDNA genes may be misleading for phylogenetic inferences, because they can produce artefacts such as Long Branch Attraction (LBA), when distantly related taxa with similar base composition tend to group together, or the opposite, Long Branch Repulsion (LBR), when closely related taxa with reverse strand-specifi c biases do not group together (Hassanin et al. 2005; Hassanin 2006; Arabi et al. 2010, 2012).

Here, we suggest that the CO1 polyphyly of Achelinae and Ammotheidae can be explained by both LBA and LBR artefacts, because two groups of Achelinae have very different base compositions, and because each of them tends to be attracted by the taxa with the most similar strand-specifi c bias, i.e., Endeidae for the “Achelia sawayai group” and Pallenopsis macneilli Clark, 1963 for all other species of Achelinae, resulting in the apparent polyphyly of Achelinae and Ammotheidae. This effect is particularly misleading for Ammotheidae because their diversifi cation is assumed to be ancient, as revealed by their remarkable morphological diversity and by our analyses of 18S and CO1 genes (up to 25% divergence between ammotheid CO1 sequences). In this context, and given that the 18S gene is more appropriate for studying the deepest nodes of classes (e.g., Mallatt et al. 2004; Arabi et al. 2012), we conclude that our nuclear gene gave accurate information for the monophyly of Ammotheidae, Ammotheinae and Achelinae. Morphologically, all representatives of Ammotheidae sensu Bouvier (1923) share the structure of the ovigers with feeble strigilis, without rows of compound spines, and generally without terminal claw. The grouping of Achelia, Ammothella s. str., Nymphopsis and Tanystylum in the subfamily Achelinae also makes sense from a purely morphological perspective. Indeed, Ammothella s. str. and Nymphopsis, which are shown as sister-groups in our study, share a lot of characters (long abdomen, eventually bi-articulated and trumpet-like scapes, long ocular tubercle), while Achelia and Tanystylum share a discoidal body outline. Taxonomic implications Only two ammotheid genera, Nymphopsis and Sericosura, are found to be monophyletic. Interestingly, specimens of Sericosura from the Pacifi c (Sericosura sp. 1 and S. venticola Child, 1987) occupy a paraphyletic position with respect to the Atlantic specimens (Sericosura sp. 2 and S. heteroscela Child & Segonzac, 1996). This pattern is coherent with the biogeographic model proposed by Bachraty et al. (2009), in which they suggested that most hydrothermal vent taxa have dispersed from the Pacifi c into the North Atlantic Ocean by a deep-sea corridor that stayed open until the closure of the Panama Isthmus around 3 million years ago.

The genus Achelia is polyphyletic: most species are grouped into a robust clade, whereas the “Achelia sawayai group” is placed within the genus Tanystylum. However, the paraphyly of Tanystylum was not highly supported by the 18S dataset, suggesting that the hypothesis of monophyly cannot be excluded. Although Achelia and Tanystylum share some superfi cial similarities, the latter genus shows a typical morphology characterized by palps with a reduced number of articles. Therefore, we consider that

15 European Journal of Taxonomy 286: 1–33 (2017) further studies are needed to decide whether a new genus should be described for members of the “Achelia sawayai group”. The inclusive position of Acheliana within the genus Ammothea suggests that Acheliana should be synonymized with Ammothea. This result was partially perceived by Arnaud (1971a), who noted the close relationship between the two genera. However, the type species of Acheliana, A. tropicalis Arnaud, 1971, must be re-examined to provide a defi nitive conclusion. The genus Cilunculus is polyphyletic in the 18S tree, because the species C. gracilis is included within Ammothella. Misidentifi cation is rather unlikely, as the 18S sequence of C. gracilis was produced by Nakamura et al. (2007), who described the species with Child in 1991 (Nakamura & Child 1991). A taxonomic issue is a more plausible explanation. Indeed, C. gracilis is one of the seven species of Cilunculus presenting a two-jointed scape, a characteristic shared with all species of the genus Ammothella. Nakamura & Child (1991) themselves recognized that C. gracilis was closely allied to Ammothella. Its assignation to the genus Cilunculus was based on the presence of a (shallow) hood-like extension of the head above the chelifores and the proboscis (Loman 1908). However, according to Child (1994), this is the only character to “temporarily” support a genus (Cilunculus) which “hangs rather precariously over the pit of synonymy”, and Nakamura et al. (2007) also expressed the unclearness of this character for several species. Now, it is becoming obvious that a revision of the genus Cilunculus is urgently needed, as our results confi rmed that the presence of a cephalic hood is not a reliable character for diagnosing members of this taxon. As there is no reason to wait to reassign the currently discussed species to the genus Ammothella, we refer to it as Ammothella gracilis (Nakamura & Child, 1991) comb. nov. The status of other species of Cilunculus with two-jointed scapes and shallow hoods will probably follow the same reassignment in forthcoming revisions.

The genus Ammothella is polyphyletic in all analyses, as most species are clustered into the Achelinae, whereas A. stauromata, and potentially A. biunguiculata (according to 18S data), are robustly placed in the Ammotheinae. From a morphological point of view, both latter species possess the typical characters of the genus Ammothella, but A. biunguiculata shows a very original profi le with short chelifores and short abdomen (see Dohrn 1881; Bouvier 1923; Hedgpeth 1941), and A. stauromata is easily recognizable, with its characteristic dorso-median tubercles (see Child 1982; Fig. 6A, C). Therefore, both molecular and morphological data suggest that the taxonomic status of these two species must be revised. For A. biunguiculata, no taxonomic change can be proposed here because we do not have morphological material and the CO1 sequence is not available. For A. stauromata, however, we can describe a new genus, maybe provisional, as there is no evidence for a relationship with any other described genus. Thus, this taxon must receive due attention in future studies.

Family Ammotheidae Dohrn, 1881 Subfamily Ammotheinae Dohrn, 1881 emend. Teratonotum gen. nov. urn:lsid:zoobank.org:act:CD6D82C7-E0EC-4E46-A2C6-559678E48B29 Fig. 6

Type species Ammothella stauromata Child, 1982: 270 (in list), 271–273, fi g. 1.

Ammothella stauromata – Arango 2003a: 2730–2731. — Bamber 2004: 2–3, 21 (in tab.); 2007a: 256 (in list). — Child 1987: 180 (in list); 1988: III (in list), 5–7, 30–31 (in list); 1990: 316 (in description); 1996: 544; 1998: 290–291. — Müller 1989: 125; 1990a: 66; 1990b: 106. — Nakamura & Child 1988: 809–810. — Stock 1994: 29.

16 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

Fig. 6. Teratonotum stauromatum (Child, 1982) gen. et comb. nov. (MNHN-IU-2013-17964). A. Dorsal view. B. Propodus of third leg. C. Dorsal view of body. D. Ventral view of body. Abbreviations: ab = abdomen; ac = auxiliary claw; ch = chelifore; ct = chelifore tubercle on the anterior tip of the fi rst scape; dt = dorsal tubercle; mc = main claw; ot = ocular tubercle; ov = oviger; pa = palp; pb = bulbous tubercle bearing the palp; pp = propodus; pr = proboscis; s = strigilis; t = tarsus. Scale bars: A = 0.5 mm; B = 0.1 mm; C–D = 0.2 mm.

17 European Journal of Taxonomy 286: 1–33 (2017)

Diagnosis Trunk slender, totally segmented; ocular tubercle present, with three long, slender dorso-median tubercles; abdomen long, straight, almost vertical; scapes bi-articled, more proximal article with dorso- distal slender tubercle, chelae atrophied in adult; palps 9-articulated, originating from bulbous tubercles (with slender tubercle) placed anterolaterally on cephalic segment; oviger 10-articulated, originating ventral to fi rst lateral processes, strigilis with denticulate spines; third leg with a single cement tube at anterior tip in males, tarsus short, propodus curved, auxiliary claws present. Etymology τέρας (téras): monster, and νῶτον (nōton): back (ancient Greek); referring to its remarkable back tubercles. Gender neutral.

An underestimated biodiversity The CO1 data generated in this study on specimens collected during the recent MNHN expeditions indicate that the diversity of Ammotheidae was poorly represented in the nucleotide databases, such as GenBank and BOLD (Ratnasingham & Hebert 2007). Indeed, all our new CO1 sequences show at least 7% of nucleotide divergence with the ca 430 pycnogonid sequences available in GenBank, and even up to 11% if the genus Sericosura is excluded from the comparisons. This means that none of the ammotheids analysed here can be identifi ed at the species level using molecular barcoding. Actually, this result is not surprising if we consider that most of the pycnogonids previously registered in the GenBank and BOLD databases were collected along the coastlines of temperate South America (Chile and Argentina) and Antarctica (Fig. 1), whereas our specimens come from widely spread geographic areas, i.e., French Guiana, Guadeloupe, Madagascar, Marquesas Islands, New Caledonia, Papua New Guinea, Vanuatu, and Atlantic and Pacifi c vents (Fig. 2). Besides, another issue for molecular taxonomy is the fact that most of the barcode sequences deposited in BOLD were not identifi ed to the family level (958 of 1315, 72.85%). Beyond a problem of molecular taxonomy, ammotheids, and more generally pycnogonids, suffer from a lack of knowledge. For instance, our specimens collected along the coastlines of southern Madagascar show a far more rich diversity than previously recorded in the literature: fi ve different species of Achelia were identifi ed, whereas only two species were previously known from Madagascar; none of the species of Ammothella and Endeis studied here were known in the region; and a new species of Acheliana was found (Arnaud 1971a, 1971b, 1972, 1973; Stock 1974). In a more general way, it seems that the large collection of sea spiders assembled during the MNHN expeditions represents an important input to our knowledge of this group and promises the description of numerous new species (in prep.). Acknowledgements The studied specimens were collected during MNHN expeditions of the programs “Tropical Deep Sea Benthos” and “Planète Revisitée” (http://laplaneterevisitee.org). Deep-sea specimens were mainly collected during the many cruises organized by P. Bouchet, S. Samadi and L.C. of the MNHN and Institut de Recherche pour le Developpement (IRD). Recent expeditions were funded by the Total Foundation, the Prince Albert II of Monaco Foundation, the Sloan Foundation, the Stavros Niarchos Foundation and conducted by the MNHN and Pro-Natura International. The KARUBENTHOS expedition was sponsored via European funding (FEDER), the Port Autonome de la Guadeloupe (PAG). Specimens from hydrothermal vents were provided by the IFREMER:BICOSE and FUTUNA 3 cruises (fi nancial support was provided by the Eramet and Technip companies). The CEAMARC cruise on RSV Aurora Australis was funded by the Australian Antarctic Division, the French polar institute IPEV, CNRS and the ANR project ANTFLOCKs (G. Lecointre). Special thanks to S. Hourdez for the specimens from the Hydromar cruise, to N. Schnell for photography and to the editor and the two anonymous reviewers for useful comments on the fi rst version of the manuscript. R.S. acknowledges the French-Taiwanese

18 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae grant TF-DeepEvo co-funded by the ANR and the Ministry of Science and Technology of Taiwan for supporting his visit to Germany. This work was supported by the project “Taxonomie moléculaire: DNA Barcode et Gestion durable des Collections”, funded by the MNHN and the “Bibliothèque du Vivant” network funded by the CNRS, MNHN, INRA and CEA (Genoscope). References Arabi J., Cruaud C., Couloux A. & Hassanin A. 2010. Studying sources of incongruence in arthropod molecular phylogenies: sea spiders (Pycnogonida) as a case study. Comptes Rendus Biologies 333 (5): 438–453. http://dx.doi.org/10.1016/j.crvi.2010.01.018 Arabi J., Judson M.L., Deharveng L., Lourenço W.R., Cruaud C. & Hassanin A. 2012. Nucleotide composition of CO1 sequences in Chelicerata (Arthropoda): detecting new mitogenomic rearrangements. Journal of Molecular Evolution 74 (1–2): 81–95. http://dx.doi.org/10.1007/s00239-012-9490-7 Arango C.P. 2003a. Sea spiders (Pycnogonida, Arthropoda) from the Great Barrier Reef, Australia: new species, new records and ecological annotations. Journal of Natural History 37: 2723–2772. http:// dx.doi.org/10.1080/00222930210158771 Arango C.P. 2003b. Molecular approach to the phylogenetics of sea spiders (Arthropoda: Pycnogonida) using partial sequences of nuclear ribosomal DNA. Molecular Phylogenetics and Evolution 28 (3): 588–600. http://dx.doi.org/10.1016/S1055-7903(03)00069-1 Arango C.P. & Wheeler W.C. 2007. Phylogeny of the sea spiders (Arthropoda, Pycnogonida) based on direct optimization of six loci and morphology. Cladistics 23 (3): 255–293. http://dx.doi.org/10.1111/ j.1096-0031.2007.00143.x Arango C.P., Soler-Membrives A. & Miller K.J. 2011. Genetic differentiation in the circum-Antarctic sea spider Nymphon australe (Pycnogonida: Nymphonidae). Deep Sea Research II 58 (1): 212–219. http://dx.doi.org/10.1016/j.dsr2.2010.05.019 Arnaud F. 1971a. Acheliana tropicalis n. gen., n. sp., pycnogonide des récifs coralliens du sud-ouest de Madagascar. Beaufortia 18 (241): 199–204. Arnaud F. 1971b. Pycnogonides des récifs coralliens de Madagascar. 2. Redescription de Pycnogonum madagascariensis Bouvier, 1911. Téthys 1: 161–64. Arnaud F. 1972. Pycnogonides des récifs coralliens de Madagascar. 3. Famille des Callipallenidae. Téthys 3: 157–64. Arnaud F. 1973. Pycnogonides des récifs coralliens de Madagascar. 4. Colossendeidae, Phoxichilidiidae et Endeidae. Téthys 4 (4): 953–960. Arnaud F. & Bamber R.N. 1987. The biology of Pycnogonida. Advances in Marine Biology 24: 1–96. http://dx.doi.org/10.1016/S0065-2881(08)60073-5 Bachraty C., Legendre P. & Desbruyères D. 2009. Biogeographic relationships among deep-sea hydrothermal vent faunas at global scale. Deep Sea Research I 56 (8): 1371–1378. http://dx.doi. org/10.1016/j.dsr.2009.01.009 Bamber R.N. 2004. Pycnogonids (Arthropoda: Pycnogonida) from French cruises to Melanesia. Zootaxa 551: 1–27. Bamber R.N. 2007a. Pycnogonida of New Caledonia. In: Payri C.E. & Richer de Forges B. (eds) Compendium of Marine Species of New Caledonia: 255–257. Documents Scientifi ques et Techniques, deuxième édition, IRD Nouméa 117. Bamber R.N. 2007b. A holistic re-interpretation of the phylogeny of the Pycnogonida Latreille, 1810 (Arthropoda). Zootaxa 1668: 295–312.

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Manuscript received: 15 February 2016 Manuscript accepted: 29 October 2016 Published on: 24 February 2017 Guest editors: Line Le Gall, Frédéric Delsuc, Stéphane Hourdez, Guillaume Lecointre and Jean-Yves Rasplus Desk editor: Danny Eibye-Jacobsen

Printed versions of all papers are also deposited in the libraries of the institutes that are members of the EJT consortium: Muséum national d'Histoire naturelle, Paris, France; Botanic Garden Meise, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Natural History Museum, London, United Kingdom; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of Denmark, Copenhagen, Denmark; Naturalis Biodiversity Center, Leiden, the Netherlands.

22 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae 2007 2010 et al. et al. Arabi Nakamura ––– DQ390087–– KF603901 DQ389932 KF603907 KF603909 – – -Australia Victoria, – Chile, Region de los Lagos AB292185Wheeler 2007 Arango & Chile, Region de los Lagos Chile, Region de los Lagos & Melzer 2002 Weis Manazuru, Kanagawa & Melzer 2002 Weis & Melzer 2002 Weis –– FJ862875 – FJ862857 AF005438 Brittany ? Giribet & Ribera 2000 – DQ389888 DQ389888– DQ390070Antarctica Palmer S DQ389916Wheeler 2007 Arango & Colombian CaribbeanWheeler 2007 Arango & IU-2011-682IU-2011-744 KX535401IU-2011-789IU-2011-790 KX536462 KX535415 -IU-2011-791 KX535370 KX536473 KX535438 KX536436 south Madagascar – – south Madagascar south Madagascar this study south Madagascar south Madagascar this study this study this study this study IU-2011-726IU-2011-727 KX535354 KX535359 KX536425 KX536430 south Madagascar south Madagascar this study this study IU-2011-702 KX535360 KX536431 south Madagascar this study IU-2011-752 KX535355 KX536426 south Madagascar this study IU-2011-712 KX535349 KX536423 south Madagascar this study IU-2011-687 KX535419 KX536476 south Madagascar this study IU-2012-811 KX535383 KX536451 Marquesas this study IU-2012-804 KX535362 KX536433 Marquesas this study IU-2012-1221 KX535422 KX536479 Madang this study IU-2012-1244IU-2012-1269 KX535421 – – KX536478 Madang Madang this study this study IU-2013-18598 KX535449 KX536500 Kavieng this study IU-2013-17934IU-2013-18597 KX535440 KX535372 KX536493 KX536441 Kavieng Kavieng this study this study voucher sp. 1 sp. 1 sp. 3 sp. 5 sp. 7 sp. 9 sp. 9 sp. 10 sp. 10 sp. 12 AcheliaAcheliaAcheliaAchelia Achelia assimilis Achelia Achelia assimilis Achelia assimilis Achelia assimilis Achelia bituberculata AcheliaAcheliaAcheliaAcheliaAchelia Achelia boschi Achelia boschi Achelia boschi Achelia boschi Achelia echinata Achelia Achelia boschi Achelia Achelia hispida AcheliaAchelia Achelia hoeki Achelia mixta Achelia Achelia AcheliaAcheliaAcheliaAchelia Achelia mixta Achelia nana Achelia nana Achelia sawayai Achelia Achelia Achelia Achelia Achelia Achelia AcheliaAcheliaAchelia Achelia Achelia Achelia AcheliaAcheliaAchelia Achelia Achelia Achelia CO1 and 18S sequences used for this study, including GenBank accession numbers. CO1 and 18S sequences used for this study, Family Genus Species MNHN code CO1 18S Geographical origin Reference AmmotheidaeAmmotheidae – Ammotheidae gen sp IU-2013-18602 KX535450 KX536501 Iles du Salut this study Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Appendix 1.

23 European Journal of Taxonomy 286: 1–33 (2017) 2007 2007 2007 2007 2009 2009 2009 2009 2009 2010 (unpubl.) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Arabi Nielsen Nielsen Nielsen Nielsen Nielsen Nakamura Nakamura Nakamura Nakamura Tamaoki Tamaoki –– – – AB292188 AB292184 Hokkaido off Manazuru, Kanagawa – – AB292186 Shimoda, Izu, Shizuoka –– DQ390091 FJ969350 DQ389936 – California, USAWheeler 2007 Arango & Ross Sea –––– FJ969346– FJ969347 FJ969348 DQ300052 –– FJ969349 – DQ389894– – DQ390081 – FJ862871Antarctica Palmer S DQ389926 Ross Sea FJ862850 Ross Sea Ross Sea Wheeler 2007 Arango & California, USA Ross Sea ? Wheeler 2007 Arango & ––– KF603912 DQ390056 DQ389899 – – AB292060 Colombian CaribbeanWheeler 2007 Arango & Shimoda, Izu, Shizuoka Chile & Melzer 2002 Weis – – LC010686 ? IU-2011-684 KX535350 KX536424 south Madagascar this study IU-2011-659IU-2011-681 KX535416 KX535418 KX536475 – south Madagascar south Madagascar this study this study IU-2011-764 KX535400 – south Madagascar this study IU-2012-963IU-2012-898 KX535424IU-2012-972 KX535380 KX535368 – KX536438 – Guadeloupe Marquesas Guadeloupe this study this study this study IU-2012-806IU-2012-807 KX535371IU-2012-808 KX535369IU-2012-809 KX536440 KX536439 KX535378 – KX536448 KX536446 Marquesas Marquesas Marquesas Marquesas this study this study this study this study IU-2013-18601IU-2013-17914 KX535377IU-2013-18604 KX535436 KX536447 KX535428 KX536490 KX536483IU-2013-18586 KX535382 Kavieng Iles du Salut KX536450 Iles du Salut Madang this study this study this study this study )

fi sp. 5 sp. 7 sp. 7 sp. sp. sp. 1 sp. 2 sp. 6 sp. 12 sp. sp. sp. 15 sp. 15 sp. 15 sp. 15 Cilunculus gracilis Ammothella gracilis (ex AcheliaAcheliaAcheliaAcheliaAchelia Achelia Achelia Achelia Achelia Achelia superba Acheliana Acheliana AchelianaAcheliana Acheliana Acheliana Ammothea Ammothea longispina Ammothea Ammothea calmani AmmotheaAmmotheaAmmothea Ammothea carolinensis Ammothea Ammothea carolinensis Ammothea Ammothea clausi Ammothea Ammothea glacialis Ammothea hilgendor Ammothea Ammothea ovatoides Ammothea Ammothea Ammothea spinosa AmmothellaAmmothella Ammothella indica Ammothella setacea AmmothellaAmmothellaAmmothellaAmmothella Ammothella Ammothella Ammothella Ammothella Ammothella Ammothella Ammothella Ammothella Ammothella Ammothella Ammothella AmmothellaAmmothella Ammothella appendiculata Ammothella biunguiculata Ammothella Ammothella biunguiculata Family Genus Species MNHN code CO1 18S Geographical origin Reference Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae

24 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae 2007 et al. Nakamura – DQ390074 DQ389919 Colombian CaribbeanWheeler 2007 Arango & –– DQ390094 DQ389938 – AB292187 California, USA Shimoda, Izu, Shizuoka Wheeler 2007 Arango & – DQ390069 DQ389915 Colombian CaribbeanWheeler 2007 Arango & IU-2011-679 KX535385 – south Madagascar this study IU-2011-602IU-2011-603 KX535417 KX535356 KX536474 KX536427 Mozambic Channel Mozambic Channel this study this study IU-2011-703 KX535429 KX536484 south Madagascar this study IU-2011-661 KX535363 KX536434 south Madagascar this study IU-2011-676 KX535366 KX536437 south Madagascar this study IU-2011-691 KX535347 – south Madagascar this study IU-2011-692 KX535348 – south Madagascar this study IU-2011-724 KX535353 – south Madagascar this study IU-2012-816IU-2012-954 KX535443 KX535374 KX536495 KX536443 Marquesas Guadeloupe this study this study IU-2011-2583 KX535367 – Manus Island this study IU-2007-4650 KX535357 KX536428 New Caledonia this study IU-2012-1204IU-2012-1206 KX535352 KX535351 – –IU-2013-6573IU-2013-6580 KX535411IU-2013-6600 KX535414 KX535392 – Madang – Madang – Madang this study Madang this study Madang this study this study this study IU-2013-19238 KX535384 KX536452 N Medio-Atlantic Ridge, Logatchev site this study IU-2013-19237 KX535437 KX536491 N Medio-Atlantic Ridge, Logatchev site this study IU-2014-10210 KX535442 KX536494 Futuna this study IU-2014-10209 KX535439 KX536492 Futuna this study IU-2013-18621IU-2013-18639 KX535448IU-2013-18640 KX535399IU-2013-18641 KX536499 KX535393 KX536461 KX535394 –IU-2013-18608 –IU-2013-15606 Madang KX535390 Kavieng KX535444 KX536456 KX536496 Kavieng N Medio-Atlantic Ridge, Snake Pit site Kavieng this study Iles du Salut this study this study this study this study this study sp. sp. 2 sp. 14 sp. 15 sp. 2 sp. 1 sp. 1 Sericosura Sericosura Cilunculus Cilunculus sewelli Cilunculus Cilunculus sewelli Cilunculus Cilunculus scaurus CilunculusCilunculus Cilunculus armatus Cilunculus roni Sericosura Sericosura Sericosura Sericosura SericosuraSericosura Sericosura heteroscela Sericosura AmmothellaAmmothellaAmmothella Ammothella Ammothella Ammothella spinifera Ammothella Ammothella tippula Ammothella Ammothella tippula Ammothella Ammothella tuberculata Nymphopsis Nymphopsis curtiscapus Nymphopsis Nymphopsis curtiscapus Nymphopsis Nymphopsis curtiscapus Nymphopsis Nymphopsis curtiscapus Nymphopsis Nymphopsis curtiscapus Nymphopsis Nymphopsis duodorsospinosa Nymphopsis Nymphopsis muscosa NymphopsisNymphopsisNymphopsis Nymphopsis muscosa Nymphopsis Nymphopsis muscosa Nymphopsis Nymphopsis muscosa Nymphopsis Nymphopsis muscosa Nymphopsis Nymphopsis muscosa Nymphopsis Nymphopsis muscosa Nymphopsis Nymphopsis muscosa Nymphopsis muscosa Nymphopsis Family Genus Species MNHN code CO1 18S Geographical origin Reference Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae

25 European Journal of Taxonomy 286: 1–33 (2017) 2007 2007 2010 2010 (unpubl.) et al. et al. et al. et al. et al. this study this study Arabi Masta Weis & Melzer 2002 Weis Nakamura Nakamura Tamaoki Tamaoki c ventsWheeler 2007 Arango & fi Lucky Strike site Lucky Strike site N Medio-Atlantic Ridge, N Medio-Atlantic Ridge, y de la Antarctica Chilena y de la Chile, Region de Magallanes – FJ862870 FJ862849 Australia –– DQ390064 GU370074 DQ389910 –Argentina Mar del Plata, Wheeler 2007 Arango & Massachussetts – KF603971 – –––– KF603964– KF603965– KF603966 KF603967 – KF603969 – KF603970 – – – Chile, Region de los Rios – Chile, Region de los Rios Chile, Region de los Rios & Melzer 2002 Weis Chile, Region de los Rios & Melzer 2002 Weis Chile, Region de los Rios & Melzer 2002 Weis Chile, Region de los Rios & Melzer 2002 Weis & Melzer 2002 Weis & Melzer 2002 Weis –– DQ390080 DQ3900990 DQ389925 DQ389935 North Paci California, USAWheeler 2007 Arango & ––– FJ862876 DQ390070 FJ862858 DQ389905 – Salomon Islands AB292190 Colombian CaribbeanWheeler 2007 Arango & Chiba Taito-zaki, off Arabi et al. 2010 – – LC010685 ? – – AB292189 Shimoda, Izu, Shizuoka IU-2012-959 KX535420 KX536477 Marquesas this study IU-2014-8275 KX535408 KX536468 Iles du Salut this study IU-2013-18603IU-2013-18606 KX535445IU-2013-18587 KX535386IU-2013-18589 KX536497 KX535446IU-2013-18596 KX536453 KX535358IU-2013-18605 KX535379 KX536429 KX535396 – KX536449 Iles du Salut KX536458 Iles du Salut Kavieng Kavieng Kavieng Iles du Salut this study this study this study this study this study this study IU-2013-19242 KX535373 KX536442 IU-2013-19239 KX535346 KX536422 IU-2013-18591 KX535361 KX536432 Kavieng this study ) ) voucher voucher voucher voucher voucher voucher voucher sp. sp. 2 sp. 2 sp. 2 sp. 3 sp. 1 sp. 1 sp. 2 sp. 2 Ammothella stauromata Ammothella stauromata Teratonotum stauromatum stauromatum Teratonotum Teratonotum stauromatum stauromatum Teratonotum (ex (ex Sericosura Sericosura venticola Sericosura Sericosura Sericosura Sericosura Tanystylum Tanystylum TanystylumTanystylum orbiculare Tanystylum Tanystylum orbiculare Tanystylum TanystylumTanystylumTanystylum Tanystylum Tanystylum Tanystylum Tanystylum Tanystylum Tanystylum Tanystylum Tanystylum TanystylumTanystylum cavidorsum Tanystylum Tanystylum cavidorsum Tanystylum Tanystylum cavidorsum Tanystylum Tanystylum cavidorsum Tanystylum Tanystylum cavidorsum Tanystylum neorhetum Tanystylum TanystylumTanystylum californicum Tanystylum cavidorsum Tanystylum Teratonotum Teratonotum AscorhynchusAscorhynchusAscorhynchus castelli Ascorhynchus Ascorhynchus castelli Ascorhynchus castellioides Ascorhynchus cryptopygius Ascorhynchus Ascorhynchus auchenicum Ascorhynchus Ascorhynchus auchenicum Ascorhynchus Family Genus Species MNHN code CO1 18S Geographical origin Reference Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ammotheidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae

26 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae 2007 2007 2010 (unpubl.) (unpubl.) (unpubl.) (unpubl.) (unpubl.) (unpubl.) (unpubl.) (unpubl.) (unpubl.) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Arabi Nakamura Nakamura Chows Chows Chows Chows Chows Chows Tamaoki Tamaoki Tamaoki Tamaoki Tamaoki Tamaoki cWheeler 2007 Arango & fi ––– DQ390048 – DQ390077– DQ389890– DQ389922– AB700613 DQ390045 DQ390057Antarctica Palmer S DQ389887 DQ390082Antarctica Palmer S DQ389900 DQ389927 Japan Wheeler 2007 Arango & Antarctica Palmer S Wheeler 2007 Arango & Colombian CaribbeanAustralia Victoria, Wheeler 2007 Arango & Wheeler 2007 Arango & Wheeler 2007 Arango & –– DQ390055– DQ390075 DQ389897 DQ389920 DQ390092 DQ389937 Colombian Caribbean CaribbeanWheeler 2007 Arango & California, USAWheeler 2007 Arango & Wheeler 2007 Arango & – – AB790611 Japan – – AB700615 Japan – – AB700612 Japan – – AB700616 Japan – – AB700614 Japan – – LC010682 ? – – LC010683 ? – – AB292192 Kochi Bay, Tosa – – LC010684 ? – – AB292191 Shimoda, Izu, Shizuoka ––– DQ390085– DQ390059– DQ389930 DQ390083 DQ389994 DQ390054 DQ389928 DQ390090 DQ389896Australia Wales, New South Australia Victoria, DQ389929Wheeler 2007 Arango & Australia Victoria, Wheeler 2007 Arango & Australia Victoria, JapanWheeler 2007 Arango & Wheeler 2007 Arango & Wheeler 2007 Arango & – DQ390078 DQ389923 Antarctica, Polarsten Exp.Wheeler 2007 Arango & – DQ390063 DQ389909Antarctica Livingstone, Wheeler 2007 Arango & – DQ390061 DQ389907 North Paci – FJ862872 FJ862852 New Caledonia IU-2013-18652IU-2013-18653 KX535376 KX535364 KX536445 KX536435 Antarctica Antarctica this study this study IU-2013-18613 KX535388 KX536454 French Guiana this study IU-2013-18642 KX535430 KX536485 Kavieng this study lipes fi sp. EurycydeEurycydeEurycydeEurycyde Eurycyde curvata Eurycyde raphiaster Eurycyde Eurycyde spinosa Propallene longiceps Propallene Oropallene minor Oropallene Decolopoda Decolopoda australis Austrodecus glaciale Austrodecus CallipalleneCallipallene Callipallene brevirostris Callipallene novaezealandiae Stylopallene Stylopallene longicauda MeridionaleParapallene Meridionale ambigua Parapallene avida Colossendeis Colossendeis stramenti Colossendeis Colossendeis tenera Nymphonella Nymphonella tapetis Nymphonella Nymphonella tapetis Nymphonella Nymphonella tapetis Nymphonella Nymphonella tapetis Nymphonella Nymphonella tapetis Nymphonella Nymphonella tapetis Nymphonella Nymphonella tapetis AustropalleneAustropalleneAustropallene cornigera Austropallene Austropallene cornigera Austropallene cornigera Austropallene cristata Austropallene Ascorhynchus japonicus Ascorhynchus Ascorhynchus japonicus Ascorhynchus Ascorhynchus glaberrimus Ascorhynchus Ascorhynchus glaberrimus Ascorhynchus Cheilopallene Cheilopallene nodulosa Rhopalorhynchus Rhopalorhynchus Family Genus Species MNHN code CO1 18S Geographical origin Reference Austrodecidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Callipallenidae Colossendeidae Colossendeidae Colossendeidae Colossendeidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae Ascorhynchidae

27 European Journal of Taxonomy 286: 1–33 (2017) 2005 2010 et al. et al. Arabi Hassanin ––– DQ390068– DQ390088 DQ389914 DQ390072 DQ389933 DQ390089 DQ389918 DQ389934 Colombian Caribbean California, USA Colombian CaribbeanWheeler 2007 Arango & California, USAWheeler 2007 Arango & Wheeler 2007 Arango & Wheeler 2007 Arango & – DQ390050 DQ389892 Colombian CaribbeanWheeler 2007 Arango & – DQ390051 DQ389893Antarctica Palmer S Wheeler 2007 Arango & – – FJ862848–– Brittany – DQ390049 DQ390086 DQ389891 DQ390062 DQ389931 –Antarctica Palmer S Australia Victoria, Wheeler 2007 Arango & Antarctica Livingston, Wheeler 2007 Arango & Wheeler 2007 Arango & – AY731173–– – DQ390087 DQ390076 DQ389889– DQ389921 DQ390053Antarctica Palmer S Antarctica, Polarsten Exp. ? DQ389895Wheeler 2007 Arango & Wheeler 2007 Arango & Antarctica Palmer S Wheeler 2007 Arango & IU-2011-776 KX535441 – south Madagascar this study IU-2013-18550 KX535406 KX536466 Iles du Salut this study IU-2013-18554 KX535447 KX536498 Madang this study IU-2013-18556 KX535381 – Madang this study IU-2013-18634 KX535405 KX536465 Kavieng this study IU-2013-18637 KX535426 – Kavieng this study IU-2013-18638 KX535427 KX536482 Kavieng this study IU-2013-18648IU-2013-18649 KX535398 KX535397 KX536460IU-2013-18614 KX536459IU-2013-18615 KX535404IU-2013-18620 KX535387 KX536464 KX535434 Antarctica Antarctica KX536488 –IU-2013-18651 French GuianaIU-2013-18645 KX535435IU-2013-18545 KX535409 French Guiana this study KX536489 KX535402 French Guiana this study KX536469 this study – Adélie Terre this study Kavieng this study Iles du Salut this study this study this study IU-2013-18546 KX535407 KX536467 Iles du Salut this study IU-2013-18610 KX535432 KX536486 French Guiana this study sp. 2 sp. 3 sp. 1 sp. 3 sp. 4 sp. sp. sp. sp. sp. sp. Endeis Endeis australis EndeisEndeis Endeis clipeata Endeis mollis Endeis Endeis Endeis Endeis Endeis Endeis Endeis Endeis Endeis Endeis Endeis Endeis Endeis Endeis spinosa Endeis Endeis spinosa NymphonNymphonNymphonNymphon Nymphon articulare Nymphon brevicaudatum NymphonNymphon Nymphon hamatum Nymphon Nymphon surinamense Nymphon surinamense Nymphon surinamense Nymphon unguiculatum Nymphon Nymphon articulare PallenopsisPallenopsisPallenopsis Pallenopsis macneilli Pallenopsis macronyx Pallenopsis PallenopsisPallenopsisPallenopsis Pallenopsis Pallenopsis Pallenopsis Pallenopsis Pallenopsis Pentanymphon Pentanymphon antarcticum AnoplodactylusAnoplodactylus Anoplodactylus viridintestinalis Anoplodactylus Anoplodactylus batagensis Anoplodactylus erectus Anoplodactylus Anoplodactlylus californicus Family Genus Species MNHN code CO1 18S Geographical origin Reference Endeidae Endeidae Endeidae Endeidae Endeidae Endeidae Endeidae Endeidae Endeidae Endeidae Endeidae Pallenopsidae Pallenopsidae Pallenopsidae Pallenopsidae Pallenopsidae Pallenopsidae Pallenopsidae Nymphonidae Nymphonidae Nymphonidae Nymphonidae Nymphonidae Nymphonidae Nymphonidae Nymphonidae NymphonidaeNymphonidae – Nymphonidae gen. sp. IU-2014-8371 KX535433 KX536487 Iles du Salut this study Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae

28 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae 2010 2004 2002 2010 2005 et al. et al. et al. et al. et al. Cook Arabi Lavrov Mallatt Braband – JN018215 JN018311 – ––– NC003057– NC002629 – – NC014273 – AF000773 – – – – – Lavrov et al. 2000a Lavrov et al. 2000b Giribet & Ribera 1998 –––– – NC007444 – NC003344 AF342794 – L81946 – – – – – 2002 Winchell Mallatt & Abele 1998 Spears & Swinstrom 2009 – – L81949 –Abele 1998 Spears & – – AY210829 – – NC006079 – – – DQ390058 DQ389902 California, USAWheeler 2007 Arango & – – AF144219 –Abele 2000 Spears & – DQ390095 DQ389939 Antarctica, Polarsten Exp.Wheeler 2007 Arango & – DQ390079 DQ389924 Antarctica, Polarsten Exp.Wheeler 2007 Arango & IU-2014-8292 KX535423 KX536480 Iles du Salut this study IU-2013-18611 KX535412 KX536471 French Guiana this study IU-2013-18654 KX535375 KX536444 Vanuatu this study IU-2013-18544IU-2013-18609 KX535391 KX535413 KX536457 KX536472 Iles du Salut French Guiana this study this study IU-2013-18632IU-2013-18636 KX535403IU-2013-18643 KX535425IU-2013-18644 KX536463 KX535395IU-2013-18646 KX536481 KX535389IU-2013-18647 KX535410 KX536455 KX535431 – KX536470 Kavieng Kavieng – Kavieng Kavieng Kavieng this study this study Kavieng this study this study this study this study sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. catus fi Triops longicaudatus Triops Triops longicaudatus Triops SquillaSquilla Squilla empusa Squilla empusa Limulus Limulus polyphemus Limulus Limulus polyphemus LithobiusLithobius Lithobius for Lithobius variegatus Orthoporus Orthoporus Thyropygus Thyropygus Peripatoides Peripatoides novaezealandiae Pycnogonum Pycnogonum tuberculatum Pentapycnon Pentapycnon charcoti Pycnogonum Pycnogonum stearnsi Pycnogonum Pycnogonum diceros Opisthopatus Opisthopatus cinctipes Anoplodactylus Anoplodactylus AnoplodactylusAnoplodactylus Anoplodactylus Anoplodactylus AnoplodactylusAnoplodactylusAnoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Anoplodactylus Mastigoproctus giganteus Mastigoproctus Family Genus Species MNHN code CO1 18S Geographical origin Reference OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP OUTGROUP Pycnogonidae Pycnogonidae Pycnogonidae Pycnogonidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae Phoxichilidiidae

29 European Journal of Taxonomy 286: 1–33 (2017)

References for Appendix 1:

Arabi J., Cruaud C., Couloux A. & Hassanin A. 2010. Studying sources of incongruence in arthropod molecular phylogenies: sea spiders (Pycnogonida) as a case study. Comptes Rendus Biologies 333 (5): 438–453. http://dx.doi.org/10.1016/j.crvi.2010.01.018 Arango C.P. & Wheeler W.C. 2007. Phylogeny of the sea spiders (Arthropoda, Pycnogonida) based on direct optimization of six loci and morphology. Cladistics 23 (3): 255–293. http://dx.doi.org/10.1111/ j.1096-0031.2007.00143.x Braband A., Cameron S.L., Podsiadlowski L., Daniels S.R. & Mayer G. 2010. The mitochondrial genome of the onychophoran Opisthopatus cinctipes (Peripatopsidae) refl ects the ancestral mitochondrial gene arrangement of Panarthropoda and Ecdysozoa. Molecular Phylogenetics and Evolution 57 (1): 285–292. http://dx.doi.org/10.1016/j.ympev.2010.05.011 Cook C.E., Yue Q. & Akam M. 2005. Mitochondrial genomes suggest that hexapods and crustaceans are mutually paraphyletic. Proceedings of the Royal Society of London B 272 (1569): 1295–1304. http:// dx.doi.org/10.1098/rspb.2004.3042 Giribet G. & Ribera C. 1998. The position of arthropods in the kingdom: a search for a reliable outgroup for internal arthropod phylogeny. Molecular Phylogenetics and Evolution 9 (3): 481–488. http://dx.doi.org/10.1006/mpev.1998.0494 Giribet G. & Ribera C. 2000. A review of arthropod phylogeny: new data based on ribosomal DNA sequences and direct character optimization. Cladistics 16 (2): 204–231. http://dx.doi. org/10.1111/j.1096-0031.2000.tb00353.x Hassanin A., Léger N. & Deutsch J. 2005. Evidence for multiple reversals of asymmetric mutational constraints during the evolution of the mitochondrial genome of Metazoa, and consequences for phylogenetic inferences. Systematic Biology 54 (2): 277–298. Lavrov D.V., Boore J.L. & Brown W.M. 2000a. The complete mitochondrial DNA sequence of the horseshoe crab Limulus polyphemus. Molecular Biology and Evolution 17 (5): 813–824. http://dx.doi. org/10.1093/oxfordjournals.molbev.a026360 Lavrov D.V., Brown W.M. & Boore J.L. 2000b. A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forfi catus. Proceedings of the National Academy of Sciences 97 (25): 13738–13742. http://dx.doi.org/10.1073/pnas.250402997 Lavrov D.V., Boore J.L. & Brown W.M. 2002. Complete mtDNA sequences of two millipedes suggest a new model for mitochondrial gene rearrangements: duplication and nonrandom loss. Molecular Biology and Evolution 19 (2): 163–169. http://dx.doi.org/10.1093/oxfordjournals.molbev.a004068 Mallatt J. & Winchell C.J. 2002. Testing the new animal phylogeny: fi rst use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes. Molecular Biology and Evolution 19 (3): 289–301. http://dx.doi.org/10.1093/oxfordjournals.molbev.a004082 Mallatt J. M., Garey J.R. & Shultz J.W. 2004. Ecdysozoan phylogeny and Bayesian inference: fi rst use of nearly complete 28S and 18S rRNA gene sequences to classify the arthropods and their kin. Molecular Phylogenetics and Evolution 31 (1): 178–191. http://dx.doi.org/10.1016/j.ympev.2003.07.013 Masta S.E., McCall A. & Longhorn S.J. 2010. Rare genomic changes and mitochondrial sequences provide independent support for congruent relationships among the sea spiders (Arthropoda, Pycnogonida). Molecular Phylogenetics and Evolution 57 (1): 59–70. http://dx.doi.org/10.1016/j.ympev.2010.06.020 Nakamura K., Kano Y., Suzuki N., Namatame T. & Kosaku A. 2007. 18S rRNA phylogeny of sea spiders with emphasis on the position of Rhynchothoracidae. Marine Biology 153 (2): 213–223. http:// dx.doi.org/10.1007/s00227-007-0803-0

30 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

Nielsen J.F., Lavery S. & Lörz A.N. 2009. Synopsis of a new collection of sea spiders (Arthropoda: Pycnogonida) from the Ross Sea, Antarctica. Polar Biology 32 (8): 1147–1155. http://dx.doi.org/10.1007/ s00300-009-0611-8 Spears T. & Abele L.G. 1998. Crustacean phylogeny inferred from 18S rDNA. In: Fortey R.A. & Thomas R.H. (eds) Arthropod Relationships: 169–187. Springer Netherlands, Dordrecht. http://dx.doi. org/10.1007/978-94-011-4904-4_14 Spears T. & Abele L.G. 2000. Branchiopod monophyly and interordinal phylogeny inferred from 18S ribosomal DNA. Journal of Crustacean Biology 20 (1): 1–24. http://dx.doi.org/10.1163/20021975- 99990012 Swinstrom K. 2009. The fi rst complete mitochondrial genome sequences for stomatopod crustaceans: implications for phylogeny. Lawrence Berkeley National Laboratory. Available from http://escholarship. org/uc/item/4s4023bb [accessed 1 February 2017]. Weis A. & Melzer R.R. 2012. How did sea spiders recolonize the Chilean fjords after glaciation? DNA barcoding of Pycnogonida, with remarks on phylogeography of Achelia assimilis (Haswell, 1885). Systematics and Biodiversity 10 (3): 361–374. http://dx.doi.org/10.1080/14772000.2012.716462

31 European Journal of Taxonomy 286: 1–33 (2017)

Appendix 2A. Bayesian tree of Pycnogonida based on 135 CO1 sequences (un-partitioned analysis). Coloured rectangles show non-ammotheid families, and coloured branches discriminate ammotheid genera. The numbers at the nodes indicate the posterior probabilities superior to 0.5. Asterisks associated to taxon names indicate holotype specimens. Outgroups were removed for better readability.

32 SABROUX R. et al., Biodiversity and phylogeny of Ammotheidae

Appendix 2B. Bayesian tree of Pycnogonida based on 135 18S sequences. Coloured rectangles show non-ammotheid families, and coloured branches discriminate ammotheid genera. The numbers at the nodes indicate the posterior probabilities superior to 0.5. Asterisks associated to taxon names indicate holotype specimens. Outgroups were removed for better readability.

33